Fig 2: miR3 function in BCR signaling regulation.(a) BJAB cells were transduced with miRNA or shSYK-encoding lentivirus, followed by stimulation with anti-IgM at indicated concentrations for 2 days. CD25 expression level (mean fluorescence intensity, MFI) was analyzed by flow cytometry. (b) miR3 suppresses NF-κB activation in B cell. BJAB cells with miRNA or shRNA targeting BCR factor were treated with indicated B cell stimulants for 24 hours. Relative NF-κB activity was analyzed by 6 × κB luciferase assay (n = 3). (c) Depletion of BCR signaling factors suppresses B cell lymphoma cell proliferation. SUDHL8 cells were treated with shRNA-expressing lentivirus, followed by long-term culture in complete medium. Results as percentage of each Venus-positive population transduced with shRNA series are plotted. Similar result was observed in SUDHL4 cells (data not shown). We confirmed that the knockdown efficiencies were comparable among the shRNAs (mean fold change vs. shCtrl is 0.255 ± 0.09 in 293T cells). (d) Results of qRT-PCR in DLBCL cohort (total, n = 68; GC, n = 31; non-GC, n = 35) shown as value relative to normal B cells. (e) Western blots showing total and phosphorylated Akt and PIK3CD (p110δ) levels in SUDHL8/miRNA cells. (f) Western blots showing cellular levels of RasGRP3, Ras-GTP (active form of Ras), total Ras, phosphorylated and total Erk1/2 and Akt in SUDHL8/shRasGRP1, /shRasGRP3, /miR-200c, and /miR-203 cells.

Fig 3: Changes in subcellular distribution of RasGRP3 and PKCδ in response to treatment with PMA or I3A.HEK-293 cells expressing GFP-RasGRP3 were treated for 30 minutes with PMA or I3A at the indicated concentrations. Cell sonicates were subjected to subcellular fractionation and the levels of PKCδ and RasGRP3 were analyzed by immunoblotting. For RasGRP3, expression was detected both with an anti-RasGRP3 antibody and with an antibody against the GFP tag. Bars indicate quantitation (mean ± SEM) of the results from three independent experiments. A representative immunoblot is illustrated.

Fig 4: Effects of Citri Reticulatae Pericarpium extract (CRE) on RasGRP3 expression in Raw264.7 cells. Raw264.7 macrophages were treated with 100 μg/mL of CRE 1 h before LPS treatment at a concentration of 1 ng/mL (A) or 100 ng/mL (B). After 18 h post-LPS-treatment, the expression of RasGRP3 was determined using immunoblotting. # p < 0.05 ### p < 0.001 compared with the blank control group; and ** p < 0.01 compared with the LPS-treated group.

Fig 5: Effects of Citri Reticulatae Pericarpium extract (CRE) on the RasGRP3 expression in Raw264.7 cells transfected with RasGRP3 siRNA. Raw264.7 macrophages with or without RasGRP3 siRNA were treated with 100 μg/mL of CRE 1 h before LPS treatment at a concentration of 1 ng/mL (A) or 100 ng/mL (B). After 18 h post-LPS-treatment, the expression of RasGRP3 was determined using immunoblotting. Different letters (a–d) represent statistically significant differences (p < 0.05) (C,D).